Fostering Responsible Research with CRISPR-Cas9montoliu/CRISPR/ethics/CRISPR... · RNA and, to a...

Fostering Responsible

Research with CRISPR-Cas9

External European Experts Meeting

Paris, 13 november 2017

Setting the stage: Where do we stand

today with CRISPR technology

• The generic name should be CRISPR

• Let’s use CRISPR tools, or CRISPR

technology, or CRISPR systems, or

CRISPR-Cas, but not CRISPR-Cas9

• Many Cas-like nucleases available

(Cas9, Cpf1/Cas12a, C2c2/Cas13a,

Cas13b, etc…)

• CRISPR is the common feature

X

Homologous Recombination and Nucleases

X

with nucleases without nucleases10-1 10-4

x1000

Fixing the DSB: NHEJ vs HDR

http://www.genome.jp/kegg-bin/show_pathway?ko03450

NHEJ HDR

Genomic Editing Tools: 4 flavours

Type Sequence Homology Double Strand Break

Engineered

Meganuclease

PROTEIN

~20 to 40 nucleotides

recognition site

PROTEIN

meganuclease

Zinc-Finger Nuclease

(ZFN)

PROTEIN

1 Zinc finger (3 AA) 3 bp

PROTEIN

FokI

TALEN PROTEIN

2 AA 1 bp

PROTEIN

FokI

CRISPR-Cas9 RNA

1 ribonucleotide 1 bp

PROTEIN

Cas9

Mojica and Montoliu ( Trends in Microbiology 2016)

Prokaryotes Eukaryotes CRISPR

The CRISPR-Cas

system

The CRISPR-Cas

system

RNP

Doudna & Charpentier (2014) Science

CRISPR applications

Disrupting a gene: KO

• The easiest approach, almost trivial

nowadays

• Many similar alleles will be generated

Deletions

• Relatively straight forward, many alleles will be

generated

• INDELs at both targeting sites will be also

generated

• Inversions and other rearrangements (i.e.

duplications) can also be generated

Point mutations

• Relatively straight forward, but can be

challenging, many alleles will be generated

• INDELs will mostly be also generated

*

Knock-ins

• Most challenging approach, many protocols,

not really optimized

• Success unrelated to CRISPR, but to

endogenous repairing mechanisms (not really

working in plants)

• INDELs will mostly be also generated

Current limitations of CRISPR

• On-target uncertainty: many alleles are generated

through NHEJ

• Most/all founder edited-organisms are mosaic

• Error-prone NHEJ is the default repairing pathway

• Donor template-specific HDR is not the preferred

repairing pathway

• Off-targets: similar target sequences can be altered

• Reaching a significant number of target cells (viral &

non-viral delivery systems)

Off-target effects

On-target

effects

Mosaicism

HDR is

not the

preferred

repairing

pathway

Related to

CRISPR

Unrelated to

CRISPR

Off-targets: we can deal with them

• Off-targets depend mainly on the selected guide

RNA and, to a lesser extent, on the Cas

(different Cas have different properties)

• New algorithms developed for selecting optimal

guide RNAs (Breaking-Cas, CRISPOR, Crispr-

GOLD…)

• Can be reduced to an acceptable minimum by

reducing the amount (Cas protein, not RNA or

DNA) and the time of action of Cas nucleases

(inhibitors)

We have not found

off-target sites with

altered sequences in

genome-edited mice

Confirmed by NGS

What about off-targets?

Mostly observed in vitro

Seruggia et al. 2015 NAR

• Founder animals are

nearly always complex

mosaic

• Many different alleles

can be present

• Not all of them might

transmit through

germline

One 8-cell embryo = 16 possible alleles

On-targets: the real problem

Reference: AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACGGTAGGGTTGATTTCAGGAAATGTAA

B9040.1 AACATTGGAGGAGCTGC ACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAG-----------GTAGGGTTGATTTCAGGAAATGTAA

B9040.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGGTAGGTAGG-TTGATTTCAGGAAATGTAA

B9040.3 AACACTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAG-----------GTAGGGTTGATTTCAGGAAATGTAA

B9040.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGC-----AGGGTTGATTTCAGGAAATGTAA

B9040.5 CAAGCTCCTGCCCCACTTTCAAAGCTGTACTGAACTGCAGTTTCTTCTCCACCCAGATTCCTGCAAGACCTTGCACCGGGG------------(437bp)------------------

B9040.6 --------------------------------------------(561bp)-------------------------------------------------------------------

B9041.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCTGTTACCTAGGGTTGATTTCAGGAAATG

B9041.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAAC--------AGGGTTGATTTCAGGAAATGTAA

B9041.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAGTGTAA

B9041.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACG----GGTAGGGTTGATTTCAGGAAATGTAA

B9041.5 AACATTGGAGGAGCTGCCACTGCTATTTGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAA

B9042.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACGGTAGGGTTGATTTCAGGAAATGTAA

B9042.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAATGTAA

B9042.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAA-TGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACGGTAGGGTTGATTTCAGGAAATGTAA

B9042.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAGTGTAA

B9043.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAGTGTAA

B9043.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACACTGGTAGGGTTGATTTCAGGAAGTG

B9043.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTCCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAGTGTAA

B9043.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGATAGACATGTCGAGGA------(134bp)--------------------

B9044.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT---------------GGTAGGGTTGATTTCAGGAAATGTAA

B9044.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCGATCAGGAGTTGAGAAAAATGGTAGGTAAC--------AGGGTTGATTTCAGGAAATGTAA

B9045.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTTGTTTAT-------------------------------CAAGGTAGGGTTGATTTCAGGAAATGTAA

B9045.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAAT--------------------GGTTGATTTCAGGAAATGTAA

B9045.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTTGTTTAT-------------------------------CAAGGTAGGGTTGATTTCGGGAAATGTAA

B9046.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACCTAGGTAGGGTTGATTTCAGGAAATG

B9046.2 AACATTG--------------------------------(87bp)-------------------------------------------------TAGGGTTGATTTCAGGAAATGTAA

B9046.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCAATGGTAGGGTTGATTTCAGGAAATGTA

B9046.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCACCTAGGTAGGGTTGATTACAGGAAATG

B9060.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAA

B9060.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAGATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAA

B9060.2 GACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAA

B9060.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCT-------GTTGATTTCAGGAAATGTAA

B9060.4 AACATCGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACA----GGTAGGGTTGATTTCAGGAAATGTAA

B9064.1 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCAAAAATGGTAGGTAGGGTTGATTTCAGG

B9064.2 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTA-----------GGTAGGGTTGATTTCAGGAAATGTAA

B9064.3 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAGTGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGCAAAAATGGTAGGTAGGGTTGATTTCAGG

B9064.4 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCAGGAGTTGAGAAAAATGGTAGGTAACAGC-----AGGGTTGATTTCAGGAAATGTAA

B9064.5 AACATTGGAGGAGCTGCCACTGCTATTGGGGACCCACCAAATGTTATCATTGTTTCCAATCA------------------------------GGTAGGGTTGATTTCAGGAAATGTAA

sgRNA-A476

ssDNA

Multiple alleles present in CRISPR founder gene-edited mice

Many mutant deletion alleles generated upon targeting

the 5’ Tyr Boundary by CRISPR-Cas9

Seruggia et al. 2015 NAR

CRISPR and human embryos

• 3 studies from China using 3n/2n embryos

• Many different alleles are produced

• Most edited embryos are mosaic

• Anticipate potential off-target effects

• Need for careful risk/benefit analysis

• Consider alternative technologies (PGD: preimplantation

genetic diagnosis)

• Need to be cautious before applying

• Poses Ethics dilemas/illegal (art 13, Oviedo Convention)

• Mitalipov study (Nature, 2017). Embryos generated ad

hoc. Combined ICSI and CRISPR tolos trigger HDR without

donor DNA. Reduced mosaicism and off-targets. To review.

CRISPR-Cas9 and in vivo somatic gene therapy

CRISPR tools and somatic gene therapy

of human rare monogenic diseases

Adeno Associated Virus

AAVAAV

1

hCas9

AAV

2

sgRNA donor

Different serotypes

with diverse tropism

to different cellular types

Cargo ~4.7 kb

Flyn et al. (2015) Experimental Hematology

CRISPR-mediated

gene therapy of a

human rare disease:

chronic granulomatous

disease (CGD) in

human iPS cells

Challenge:

multiple alleles are

generated

Gene Drive: Mutagenic Chain Reaction

Gantz & Bier (Science, 2015)

Gene Drive: Mutagenic Chain Reaction

Mendelian

Non-Mendelian

The paradigm of genetic selection/improvement

High milk production

Sensitive to disease X

Low milk production

Resistance to disease X

X

High milk production

Resistance to disease X

High milk production

Sensitive to disease X

Low milk production

Resistance to disease X

X

Intermediate milk production

Intermediate susceptibility to disease X

The paradigm of genetic selection/improvement

The paradigm of genetic selection/improvement

High milk production

Sensitive to disease X

Low milk production

Resistance to disease X

Gene X

Transgenesis

High milk production

~Sensitive to disease XLow expression of transgene

Chromosomal position effects

The paradigm of genetic selection/improvement

High milk production

Sensitive to disease X

Low milk production

Resistance to disease X

Gene X

A T

Gene X

C G

Gene Editing

CRISPR-Cas9

The paradigm of genetic selection/improvement

High milk production

Sensitive to disease X

Low milk production

Resistance to disease X

Gene X

A T

Gene X

A T

High milk production

Resistance to disease X

transgene+ transgene GMO ✓

Gene X

A

Gene X

T

versus Genetic variants/Breeds

GMOX

Gene X

A

Gene X

T

Gene-edited organism

GMO ?

CRISPR

Gene-edited organisms has to be considered GMO?

A gene-edited organism is NOT a GMO

www.cnb.csic.es/~montoliu/CRISPR/

Google for CNB + CRISPR

The CRISPR web page at CNB